The present invention relates generally to inhibitors of viral proteases, and particularly to certain peptide methyl ketones useful as specific inhibitors of protein processing by picornavirus proteases.
Proteases are enzymes which cleave proteins at specific peptide bonds. In living systems, highly specific proteases and complementary protease inhibitors mediate or control a broad spectrum of biological functions. For example, proteases cleave precursors to form active proteins in post-translational processing of polypeptides, provide mechanisms for zymogen activation cascade reactions such as blood coagulation, fibrinolysis, and complement reactions of the immune system, and mediate transport of selected proteins across biological membranes. Accordingly, proteases represent potential targets for therapeutic agents designed to function as specific inhibitors of protease activity.
Proteases encoded by viral genomes play a critical role in viral reproduction. Viral proteases cleave large precursor polypeptides produced by infected cells into smaller protein components, or subunits, which are subsequently assembled to form functional virus structures. Lozitskii et al., Usp. Sovrem. Biol. 93:352-362 (1982), discuss the role of proteolysis in reproduction of avian and mammalian viruses, and have surveyed part of the literature relating to viral protease inhibitors.
Picornaviruses represent a significant class of viral pathogens in humans and other mammals. Included within this class are polioviruses, rhinoviruses, and the viruses which are the etiologic agents of hepatitis A and hoof-and-mouth disease. During picornavirus replication, viral mRNA is translated in a continuous passage of ribosomes along a viral mRNA molecule, producing a linear protein product which is cleaved at selected sites by virus-specified proteases prior to dissociation of a protein/ribosome complex.
A number of workers have sought specific inhibitors of picornavirus protease activity. Korant, J. Virol. 10:751-759 (1972), discloses inhibition of poliovirus and echovirus-12 protein processing by chloromethyl ketone derivatives of simple amino acids. Specifically, Korant discloses inhibition by tolylsulfonylphenylalanyl chloromethyl ketone (TPCK) and tolylsulfonyllysyl chloromethyl ketone (TLCK). Summers et al., J. Virol. 10:880-884 (1972), similarly disclose inhibition of protease cleavage of large poliovirus-specific polypeptides by TPCK, TLCK, and D- and L-isomers of carbobenzyloxyphenylalanyl chloromethyl ketone (ZPCK). In a subsequent report, Korant et al., Proc. Natl. Acad. Sci. USA 76:2992-2995 (1979), describe inhibition of poliovirus protein processing by carbobenzyloxyleucyl chloromethyl ketone (ZLCK).
Various peptide derivatives with capacity to inhibit protease activity are known. Powers, "Haloketone Inhibitors of Proteolytic Enzymes", in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein, ed., (Marcel Dekker, New York, 1977), pp. 65-178, has surveyed the literature relating to inhibition of protease activity by haloketone derivatives of amino acids and peptides. Powers et al., Biochim. Biophys. Acta 480:246-261 (1977), disclose inhibition of subtilisin BPN', a bacterial protease, by a species of peptide chloromethyl ketones. Of the compounds tested, acetyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl chloromethyl ketone (Ac-Phe-Gly-Ala-LeuCH.sub.2 Cl) was the fastest inhibitor. A related compound, methoxysuccinyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl chloromethyl ketone (MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl) is disclosed by Enzyme Systems Products in a November 1981 product bulletin.
Ito et al., Biochem. Biophys. Res. Commun. 49:343-349 (1972), describe experiments involving inhibition of chymotrypsin, a digestive protease, by certain peptide aldehydes. Ito et al. also tested for inhibition of chymotrypsin by Ac-Leu-Leu-PheCH.sub.3, a tripeptidyl methyl ketone. However, no inhibition was observed at an inhibitor concentration of 600 .mu.g/mL.
Finally, Fittkau et al., "Synthesis and Properties of Peptide Ketones", in Peptides 1982, Blaha et al., eds., (de Gruyter, New York, 1983) pp. 617-622, disclose inhibition of thermitase, a thermostable serine protease of Thermoactinomyces vulgaris, by certain peptide methyl ketones.
It has now been found that a selected group of peptide methyl ketones are inhibitors of picornavirus protease activity. These compounds represent possible therapeutic agents for treatment of viral infection in mammals.